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. 2015 Dec 1;31(12):1278–1296. doi: 10.1089/aid.2015.0074

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Copyright 2015, Mary Ann Liebert, Inc.

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FIG. 5. — Nef expression and function are preserved in A3R5.7 cells and PBMCs infected with LucR.6ATRi reporter virus. (A–C) Evaluation of expression (A, B) and function (C) of Nef in the T cell line A3R5.7 and PBMCs after infection with 293T transfection-derived (TD) LucR.T2A reporter (NL-LucR.T2A-BaL.ecto), LucR.6ATRi reporter (NL-LucR.6ATRi-BaL.ecto), and nonreporter (NL-BaL.ecto) viruses. Mutations that abrogate Nef expression (Nef^STOP) were incorporated into each background. An abrogated Nef myristoylation mutant (Nef^G2A) was also included in the NL-BaL.ecto background. Unaltered nonmutated (wild-type) Nef is indicated as “Nef.” A3R5.7 cells or PBMCs infected with HIV-1 were identified by intracellular p24 staining in order to normalize the number of infected cells analyzed by Western blot, and for gating on HIV-1-infected (p24⁺) cells for flow cytometric analysis. (A, B) Western blot evaluation of Nef (lower panels, with anti-Nef mAb clone EH1) and LucR (upper panel) levels in A3R5.7 cells (A) or PBMCs (B) 96 hpi with TD LucR.T2A or LucR.6ATRi reporter and nonreporter viruses. For the evaluation of Nef, original and digitally enhanced images are presented in parallel to illustrate significantly reduced (A) or absent (B) Nef levels (see closed arrowheads, ▲). Replica blots were prepared in parallel and probed with anti-p24 (Gag) (middle panel) to demonstrate loading of equivalent numbers of infected cells. (C) Flow cytometric evaluation of Nef function, as measured by MHC-I down-modulation on infected (p24⁺) A3R5.7 cells (left panels) or PBMCs (right panels) 96 hpi. Levels of MHC-I down-regulation for NL-LucR.T2A-BaL.ecto and NL-LucR.6ATRi-BaL.ecto were compared to both nonreporter virus (NL-BaL.ecto), containing wild-type Nef (filled histograms), and either NL-LucR.T2A-BaL.ecto or NL-LucR.6ATRi-BaL.ecto containing the abrogated Nef expression mutation (Nef^STOP) (dashed histogram). Additionally, levels of MHC-I down-regulation for nonreporter virus encoding the Nef^G2A mutation were compared to nonreporter virus containing wild-type Nef (filled histograms) and the Nef^STOP mutation (dashed histogram).